Dynein structure and power stroke

Dynein ATPases are microtubule motors that are critical to diverse processes such as vesicle transport and the beating of sperm tails; however, their mechanism of force generation is unknown. Each dynein comprises a head, from which a stalk and a stem emerge. Here we use electron microscopy and image processing to reveal new structural details of dynein c, an isoform from Chlamydomonas reinhardtii flagella, at the start and end of its power stroke. Both stem and stalk are flexible, and the stem connects to the head by means of a linker approximately 10 nm long that we propose lies across the head. With both ADP and vanadate bound, the stem and stalk emerge from the head 10 nm apart. However, without nucleotide they emerge much closer together owing to a change in linker orientation, and the coiled-coil stalk becomes stiffer. The net result is a shortening of the molecule coupled to an approximately 15-nm displacement of the tip of the stalk. These changes indicate a mechanism for the dynein power stroke

Direct Observation of the Myosin Va Recovery Stroke That Contributes to Unidirectional Stepping along Actin

Myosins are ATP-driven linear molecular motors that work as cellular force generators, transporters, and force sensors. These functions are driven by large-scale nucleotide-dependent conformational changes, termed “strokes”; the “power stroke” is the force-generating swinging of the myosin light chain–binding “neck” domain relative to the motor domain “head” while bound to actin; the “recovery stroke” is the necessary initial motion that primes, or “cocks,” myosin while detached from actin. Myosin Va is a processive dimer that steps unidirectionally along actin following a “hand over hand” mechanism in which the trailing head detaches and steps forward ∼72 nm. Despite large rotational Brownian motion of the detached head about a free joint adjoining the two necks, unidirectional stepping is achieved, in part by the power stroke of the attached head that moves the joint forward. However, the power stroke alone cannot fully account for preferential forward site binding since the orientation and angle stability of the detached head, which is determined by the properties of the recovery stroke, dictate actin binding site accessibility. Here, we directly observe the recovery stroke dynamics and fluctuations of myosin Va using a novel, transient caged ATP-controlling system that maintains constant ATP levels through stepwise UV-pulse sequences of varying intensity. We immobilized the neck of monomeric myosin Va on a surface and observed real time motions of bead(s) attached site-specifically to the head. ATP induces a transient swing of the neck to the post-recovery stroke conformation, where it remains for ∼40 s, until ATP hydrolysis products are released. Angle distributions indicate that the post-recovery stroke conformation is stabilized by ≥5 kBT of energy. The high kinetic and energetic stability of the post-recovery stroke conformation favors preferential binding of the detached head to a forward site 72 nm away. Thus, the recovery stroke contributes to unidirectional stepping of myosin Va

Cooperative Adaptation to Establishment of a Synthetic Bacterial Mutualism

To understand how two organisms that have not previously been in contact can establish mutualism, it is first necessary to examine temporal changes in their phenotypes during the establishment of mutualism. Instead of tracing back the history of known, well-established, natural mutualisms, we experimentally simulated the development of mutualism using two genetically-engineered auxotrophic strains of Escherichia coli, which mimic two organisms that have never met before but later establish mutualism. In the development of this synthetic mutualism, one strain, approximately 10 hours after meeting the partner strain, started oversupplying a metabolite essential for the partner's growth, eventually leading to the successive growth of both strains. This cooperative phenotype adaptively appeared only after encountering the partner strain but before the growth of the strain itself. By transcriptome analysis, we found that the cooperative phenotype of the strain was not accompanied by the local activation of the biosynthesis and transport of the oversupplied metabolite but rather by the global activation of anabolic metabolism. This study demonstrates that an organism has the potential to adapt its phenotype after the first encounter with another organism to establish mutualism before its extinction. As diverse organisms inevitably encounter each other in nature, this potential would play an important role in the establishment of a nascent mutualism in nature

Back on track – On the role of the microtubule for kinesin motility and cellular function

The evolution of cytoskeletal filaments (actin- and intermediate-filaments, and the microtubules) and their associated motor- and non-motor-proteins has enabled the eukaryotic cell to achieve complex organizational and structural tasks. This ability to control cellular transport processes and structures allowed for the development of such complex cellular organelles like cilia or flagella in single-cell organisms and made possible the development and differentiation of multi-cellular organisms with highly specialized, polarized cells. Also, the faithful segregation of large amounts of genetic information during cell division relies crucially on the reorganization and control of the cytoskeleton, making the cytoskeleton a key prerequisite for the development of highly complex genomes. Therefore, it is not surprising that the eukaryotic cell continuously invests considerable resources in the establishment, maintenance, modification and rearrangement of the cytoskeletal filaments and the regulation of its interaction with accessory proteins. Here we review the literature on the interaction between microtubules and motor-proteins of the kinesin-family. Our particular interest is the role of the microtubule in the regulation of kinesin motility and cellular function. After an introduction of the kinesin–microtubule interaction we focus on two interrelated aspects: (1) the active allosteric participation of the microtubule during the interaction with kinesins in general and (2) the possible regulatory role of post-translational modifications of the microtubule in the kinesin–microtubule interaction.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42588/1/10974_2005_Article_9052.pd

Cbfβ2 controls differentiation of and confers homing capacity to prethymic progenitors

Multipotent hematopoietic progenitors must acquire thymus-homing capacity to initiate T lymphocyte development. Despite its importance, the transcriptional program underlying this process remains elusive. Cbfβ forms transcription factor complexes with Runx proteins, and here we show that Cbfβ2, encoded by an RNA splice variant of the Cbfb gene, is essential for extrathymic differentiation of T cell progenitors. Furthermore, Cbfβ2 endows extrathymic progenitors with thymus-homing capacity by inducing expression of the principal thymus-homing receptor, Ccr9. This occurs via direct binding of Cbfβ2 to cell type-specific enhancers, as is observed in Rorγt induction during differentiation of lymphoid tissue inducer cells by activation of an intronic enhancer. As in mice, an alternative splicing event in zebrafish generates a Cbfβ2-specific mRNA, important for ccr9 expression. Thus, despite phylogenetically and ontogenetically variable sites of origin of T cell progenitors, their robust thymus-homing capacity is ensured by an evolutionarily conserved mechanism emerging from functional diversification of Runx transcription factor complexes by acquisition of a novel splice variant